ABSTRACT Interleukin 35 (IL35) has emerged as a potent immuno-suppressive cytokine in cancer, auto-immune and transplant immunology. A member of the IL12 family of cytokines, IL35 is a heterodimeric cytokine composed of the protein subunits Epstein Barr Virus Induced 3 (Ebi3) and p35 and is produced and secreted predominantly by regulatory T cells (Tregs). While IL35 has been implicated in the regulation of a number of cellular immune responses, there still exists significant debate as to whether the cytokine actually exists. This debate is predicated on the fact that attempts to isolate IL35 from blood, physiological solutions like ascites or lymph, or even cell culture supernatants have failed. We have recently immune-precipitated both Ebi3 and p35 with an antibody to the tetraspanin CD81 from mouse lymphocytes. Tetraspanins are membrane spanning proteins associated with the formation of the small, extracellular vesicles, exosomes. This interesting observation prompted us to examine whether IL35 exists as Ebi3 and p35 proteins associated with tetraspanins and secreted and acquired as an exosome. To test the idea that IL35 exists as an exosome dependent cytokine, we designed the following specific aims: Specific Aim 1: To test whether exosomes isolated from serum of tolerized groups of tetraspanin knockout mice (CD81, CD63, CD9) lose Ebi3 and p35 positive exosomes. Specific Aim 2: To examine whether lymphocytes from tolerized-CD81, CD63 or CD9 knockout mice produce and release functional IL35+ immuno-suppressive exosomes. The successful completion of the outlined specific aims in this proposal will fill a significant gap in our understanding of the cytokine IL35, and will ideally lead to future applications that further our understanding of the therapeutic implications of IL35 for humans.